The broad term goals of this project are to develop new techniques to empirically determine the mechanisms by which cells selectively differentiate and incorporate metals to take advantage of the additional steric, catalytic and redox properties these metals confer to acceptor molecules. The recent realization of the importance of Zn in life processes has underscored the need for a better understanding of the regulatory processes controlling the cytosolic free Zn2+ ion activity such that cellular processes are simultaneous protected from deficiencies and toxic excesses of this metal. One class of protein that have been implicated in metal homeostasis is metallothionein (MT). This ancient family of metal-binding proteins is ubiquitously expressed in all mammals and, in addition to Zn and Cu homeostasis, is though be involved in protecting cells against toxic metals (Cd, Hg), ionizing radiation, reactive oxygen species, electrophilic anticancer drugs and mutagens. Four major isoforms of MT exist in humans that are differentially expressed both spatially and developmentally in response to various stimuli, although their specific and interactive roles are unknown. Existing technologies allow cellular Zn transfer reactions to be monitored from only one donor. Consequently it is not known whether the metals associated with each specific isoform are kinetically and/or thermodynamically isolated from each other when concordantly expressed. This information is important in determining their individual or collective roles in metal homeostasis and detoxification. We present preliminary data on the utility of directly coupled HPLC- ICP-MS for empirically studying Zn transfer reactions between five different donor proteins. The specific aim of this project is to use this new technology to differentially label the various MT isoforms with 66/67/68/70 Zn for multiplexing transfer reactions designed to elucidate their individual and interactive roles in Zn distribution within the cell. These experiments will: (i) define Zn exchange between the specific isoforms and potential recipient apo-enzymes; (ii) determine if these transfers require ligand- ligand interaction; (iii) establish if inter-isoform exchange requires free Zn and heterodimerization via thiol bridging; (iii) utilize dual-isotopically Zn/Cd MT isoforms to study the ability of each isoform to differentially process potentially toxic Cd from Zn under varying redox conditions. [unreadable] [unreadable] Life has evolved to utilize metals and it is hard to envisage of any known cellular pathway, be it developmental, defense, cognitive, regulatory, signaling, gene activation/expression, metabolic, transport, growth etc. that does not have metals implicitly and intricately tied into it. The proposed research aims to test a novel technique that can quantitatively monitor the simultaneous movement of numerous metals between different cellular molecules. Future development of the technique and its application to proteomics will allow the study of metal-protein interactions in relatively complex samples comparable to the cytoplasmic milieu, which is an important step to understanding the regulatory role of metals in protein functioning and in cellular metabolism in vivo. [unreadable] [unreadable] [unreadable]